Postnatal development of hepatic uricase and peroxisomes in baby pigs

Comp. Biochem. Physiol. Vol. 88B, No. 3, pp. 999-1003, 1987 Printed in Great Britain

0305-0491/87 $3.00+0.00 © 1987PergamonJournals Ltd

POSTNATAL DEVELOPMENT OF HEPATIC URICASE A N D PEROXISOMES IN BABY PIGS D. GIESECI~,* A. POSI)ISCHIL,~C. LAGING~ and G. HEISS* *Institut ffir Physiologic, Physiologische Chemic und Em~hrungs-physiologie and tInstitut f~r Tierpathologie, Universit~t Mfinchen, Veterin~irstraBe 13, D-8000 Mfinchen 22 (FRG) (Tel.: 089 2180-2506) (Received 3 February 1987)

Abstract--1. The activity of uriease, the densities of peroxisomes and cores in liver samples of baby pigs up to 4 weeks of age were investigated. 2. From 1 to 4 weeks of age, uricase activity as well as counts of cores and peroxisomes increased 5.5-, 3.3- and 2-fold. 3. Uricase activity and counts of cores and peroxisomes were correlated (P < 0.001) with age in linear relationships. 4. Calculated for time of birth uricase activity was very low and ratio of cores to peroxisomes was 1: 7. 5. From 0 to 28 days of age the calculated increases of uricase activity and counts of cores and peroxisomes were 178-, 10- and 3-fold.

INTRODUCTION In purine metabolism man and anthropoid primates differ from other mammals by renal excretion of uric acid instead of allantoin. The loss of uricase (Urate oxidase EC 1.7.3.3) from liver cells is responsible for this situation which in man may result in hyperuricemia and gout (Weiner, 1979). Uric acid is also known to cause health problems in certain other mammals. Thus the Dalmatian dog excretes uric acid and is prone to urate kidney stones because of a defective transport system for uric acid in liver cells (Gieseeke and Tiemeyer, 1984). In baby pigs, pathologists have long been aware of fatal kidney infarction resulting from accumulation of crystaline uric acid (Madsen et al., 1944). The reasons for this disorder are still unexplained. In view of the increasing use of baby pigs as models in pediatric research, the problem needs more concern. According to preliminary observations (Stohrer et aL, 1985), the liver of baby pigs at 2 days of age shows no uricase activity. As the enzyme is associated with the cores of hepatic peroxisomes (Hruban and Swift, 1964), we have investigated uricase and perixosome characteristics in the liver of baby pigs during the first four weeks of life.

determination of uriease activity, the samples were immediately placed in liquid nitrogen, ground to powder and homogenized for 1 min (Ultra-Turrax Janke and Kunkel, Staufen) in Krebs-Ringer buffer solution (pH 7.4) at 3 g wet wt/50 ml. Two millilitres of the homogenate were incubated at 37°C under oxygen gas phase in 5 ml buffer medium (pH 7.4) containing various concentrations of uric acid. After 30 min, the reaction was stopped and the concentration of uric acid in the incubation mixture was determined by HPLC (Tiemeyer et aL, 1981). Uricase activity values were calculated as nkat (nmol/s) per g of liver. Morphological evaluation For morphological examination, grain-sized pieces of fiver tissue were fixed in giutaraldehyde (6.25% w/v) for 120 min and embedded according to the standard methods for transmission electron microscopy. Peroxisomes and "cores" were counted on electron micrographs of appropriate magnification by means of a semi-automatic image analyzer (Morphomat I0, Zeiss, Oberkochen). Statistics The statistical comparison of age groups by the U-test of Wilcoxon, Mann and Whitney as well as calculation of correlations and regressions were performed according to Sachs (1972).

RESULTS

MATERIALS AND METHODS Chemicals All chemicals used were of analytical reagent grade. Uric acid and other purine compounds used for analytical standardization were purchased from Sigma Chemical Co. (Miinchen). Chemicals for incubation medium were from Merck (Darmstadt), liquid nitrogen and oxygen for medical use were obtained from Linde (Miinchen). Liver samples and uricase assay Baby pigs of both sexes (German Landrace) at the age of 2-28 days were obtained from the University Farm. The animals were killed by anaesthesia (Ethomidate, Janssen, Neuss) during which liver samples were removed. For the

Basic observations

If liver homogenates from piglets were incubated with uric acid, the disappearance of substrate from the incubation medium depended largely on the age of the donor animals (Fig. 1). During the first days of postnatal life, none or only very little uricase activity could be observed. The other main factor influencing urate breakdown was substrate concentration. The effects of both substrate level and age of donor animals on uricase activity in liver homogenates are shown in Fig. 2. During the first week, enzyme activity ranged very low at all substrate levels. A marked increase was noted if samples from 999

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animals in the second and third weeks were examined, but a steep increase of uricase activity with increasing substrate concentration was only observed with liver homogenates from piglets in the fourth week. Furthermore, in samples taken during the first week, substrate levels exceeding 500 #mol/l definitely inhibited uricase activity and a similar effect was obvious in liver homogenates from baby pigs in the second week with the highest substrate level. The peroxisomes in liver cells, the site of the enzyme, were suspected to lag behind in development. These are single-membrane bound spherical bodies slightly smaller than mitochondria (Fig. 3). In most species including the pig, these intra cytoplasmatic organelles are characterized by electron-

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Fig. 2. Effect of age of baby pigs and of substrate level on uricase activity in fiver homogenates (~ + SE, N = 3-8); the age of animals was 3-6 (O), 8-13 (C)); 17-19 (ZX)and 23-28 (["1) days.

Fig. 3. Electron micrograph of liver peroxisome; core of nucleoid (arrow), marginal plate (arrow head).

dense, central rod-like structures called "cores". Furthermore, certain marginal flat areas, the so called "marginal plates", are typical morphological features of peroxisomes. The quantitative evaluation of uricase activity, peroxisomes and cores in liver samples from baby pigs at 3-28 days of postnatal life are summarized in Table 1. The four age groups represent the first, second, third and fourth week of age. From the first to the fourth week the increase of uricase activity averaged 5.5-fold. For the peroxisomes and cores, it was about 2- and 3.3-fold. Obvious differences in development were noted: uricase activity improved by a factor of 2.2 between I and 2 weeks, peroxisomes and cores only by factors of 1.2 and 1.4. The enzyme activity increased 2-fold again between 3 and 4 weeks, but only small changes occurred in peroxisomes and cores. Furthermore, the variability between animals as indicated by SD decreased from 71 to 25% for uricase activity and from 46 to 23% for pcroxisome counts between weeks 1 and 4. As for cores, the respective change was only from 58 to 46%. Relationships between uricase activity, peroxisomes and cores The increase of uricase activity with age is characterized by a linear relationship as shown in Fig. 4. The intercept on the ordinate would indicate that uricase activity at birth was only 0.11 nkat g liver. The rate of increase of uricase activity was about 2 nkat/g for every 3 days and the total increase from 0 to 28 days was about 178-fold. The density of peroxisomes increased with age in a linear progression (Fig. 5). According to this function, the peroxisome counts at birth averaged 34/3000/zm 2 and increased by about l0 for each 4 days of life or 68 for the 28 days of age examined i.e. a 3-fold increase over the initial density. Apparently, values for the individual animals were more scattered in the second half of the experimental period.

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Table I. Catalytic activity of uricase and density of peroxisomes and cores in livers of baby pigs Age (days)

Animals (N)

4.4 ± 1.3 (3-6) 10.5 ± 1.6 (8-13) 18.0 + 1.1 (17-19) 25.6 + 1.8 (23-28)

7

Uricase* (nkat/g)

Peroxisomest

3.57" ± 2.55 7.86"b ±4.00 9.49b ±3.47 19.58c ±4.89

8 8 5

Corest

45.45" ± 21.08 53.73" + 10.19 85.28b ±21.69 90.77b +25.98

14.72" ± 8.49 20.53"b + 11.59 37.69b ± 14.41 48.25b ± 21.84

*nkat uricase/g of liver wet wt (i _+SD). tCounts/3000pmz cut liver surface (i ± SD). a-c. Values in each column followed by different letters are significantly different (P < 0.01).

The effect of age of the baby pigs on the counts of peroxisomal cores is shown in Fig. 6. At birth, a mean density of 5 cores/3000 p m 2 cut liver surface could be calculated. The linear rate of increase was about 5 cores for every 3 days interval or a 10-fold total increase during the 28 days observation period. A particularly close correlation should be expected between the density of cores and the activity of uricase. The relationship (Fig. 7) was significant, but at a lower level than age-related parameters shown before. If extrapolated to zero the regression equation would indicate uricase activity of about 4 nmol/s per g when no cores were detected.

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DISCUSSION

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Eighty years ago Mendel and Mitchell (1907) failed to prove by a qualitative procedure uricase activity in fetal pig liver (about 3-4 weeks ante nature), in contrast to high activity found in livers from piglets at 2 months of age. This result has now also been confirmed for neonatal pigs as indicated by Figs 1 and 2. Obviously about 4 weeks of postnatal development are needed to achieve full uricase activity. This is in agreement with the general observation that piglets are born with a metabolically undeveloped liver (Mersmann et al., 1972; Neundorf and Seidel, 1977). This is particularly true for certain enzymes of gluconeogenesis such as glucose-6-phosphatase

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different rates. Therefore, up to 4 weeks of age, the counting of these organelles provides only limited information on the development of the uricolytic enzyme.

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activity within the first 5 days of postnatal life (Bieber et al., 1979). The results summarized in Table 1 would suggest that uricase activity in the liver of piglets is lagging behind because of the slow development of the enzyme bearing organelles, peroxisomes and cores. It has been stated (Flaks, 1971) that the microbodies (peroxisomes) of porcine hepatocytes lack distinct nucleoids (cores) and are in so far, closely similar to those of man. Figure 3 and Table 1 as well as other observations (Laging, 1985) provide sufficient evidence for the contrary. It is well known also that in the liver of the rat, maximal uricase activity is achieved within only about two weeks of postnatal life (Tsukada et al., 1968; Krahling et al., 1979). But in contrast to what is found in piglets, the enzyme is active already at 18 days of pregnancy (Tsukada et al., 1968). Furthermore, at birth the density of liver peroxisomes and core is definitely higher in the rat than in the pig (Laging, 1985). According to the regression shown in Fig. 4, uricase activity at birth (by extrapolation) is only 0.1 nkat/g liver. Similarly, Figs 5 and 6 would suggest initial numbers for peroxisomes and cores of about 34 and 5/3000/~m 2. This would mean that at this early stage, a statistical average of only every seventh peroxisome has one core. The very low level of both uricase activity and number of cores at birth is in agreement with the close association of these functional and morphological units (Hruban and Swift, 1964; Pitts and Priest, 1974). If calculated according to the regression equations in Figs 4-6, the increases between 0 and 28 days of uricase activity as well as that of densities of cores and peroxisomes would account to 178-, 10- and 3-fold, respectively. These figures suggest not only a definite increase with age of cores in peroxisomes, but in particular, a strong increase of uricase activity which is not easily explained only by the increase of cores. It has been postulated that the cores are somewhat like the morphological equivalent of uricase (Afzelius, 1965; Lata et al., 1977; Antonenkov and Panchenko, 1978). On the other hand, De Duve and Baudhuin (1966) as well as Hayashi et al. (1976) have contradicted this opinion. Our results permit the conclusion that uricase activity, counts of peroxisomes and cores in porcine liver increase with age at quite

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